Recombinant adenovirus vectors have proven to be an efficient and versatile gene therapy system for the targeted transfer of recombinant genes to diseased cells or tissue. Adenovirus vectors infect a broad range of target cells; however, high levels of gene transfer depend upon the presence of the coxsackievirus and adenovirus receptor (CAR) in the target cell. Several cell types and tissues that represent important targets for gene therapy are refractory to adenoviral infection, mainly because of low CAR expression levels (see, e.g., Havenga et al., 2001, J. Virol. 75:3335-3342). Similarly, some target cells that are readily infected by adenovirus require high levels of adenovirus particles to achieve transduction (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), exacerbating any immune response associated with adenoviral infection. Thus, considerable efforts have been directed toward increasing the efficiency of adenovirus delivery to therapeutically relevant human cells and tissues.
The cellular entry mechanism of adenovirus serotype 5 (Ad5) is composed of two separate and uncoupled steps. First, the virus binds to the host cell through a high-affinity interaction between the trimeric carboxy-terminal knob domain of the viral fiber proteins and CAR displayed on the cell surface. This primary interaction, which dictates the infectivity of the virus is followed by the association of RGD sequences in the penton base with αvβ3 and αvβ5 integrins on the cell-surface, thereby activating internalization of the virus. Strategies to alter Ad5 tropism are based on modification of the viral capsid proteins to permit the recognition of alternative cell-specific receptors. Adenovirus capsid proteins include hexon, penton base, and fiber proteins. Since the binding of the virus with its cognate cellular receptor controls the tropism of the virus, the majority of efforts have been directed at genetically modifying the carboxy-terminal knob domain of the fiber protein (for a review, see Krasnyhk et al., 2000, Mol. Ther. 1:391-405). Many laboratories have shown that it is possible to replace the Ad5 fiber gene, either partially or completely, with that of different adenovirus serotypes, thus generating a tropism derived from the donor serotype. Indeed, some of the Ad5-chimeras that have been created exhibit enhanced tropism for defined cell types. However, the flexibility of this “fiber swapping” approach is hampered by the number of serotypes available and their limited tropism. In addition, impaired viability and reduced yield of the viral chimeras have limited the exploitation of this strategy.
Recently, various labs have shown that short peptides can be incorporated at defined sites of the Ad5 fiber knob domain. The HI loop, which protrudes from the knob domain, structurally tolerates the insertion of a wide number of peptide sequences leaving fiber trimerization and CAR binding function intact. These findings prompted the screening of phage-displayed peptide libraries as a promising route to identify ligands with a desired binding specificity. The fact that ligands selected from phage libraries often do not retain their binding properties when grafted into a different protein location, i.e. the HI loop of the Ad5 fiber knob, has resulted in a paucity of successful reports using this strategy. Furthermore, insertion of these peptides can affect fiber trimerization and virus assembly.
Krasnykh et al. (1998, J. Virol. 72:1844-1852) recombinantly expressed an Ad5 fiber protein containing a FLAG octapeptide within the HI loop domain of the fiber knob. The heterologous peptide did not ablate fiber trimerization or disturb formation of the cell-binding site within the knob. Recombinant adenovirus containing said modified Ad5 fiber protein maintained proper biological function.
Dmitriev et al. (1998, J. Virol. 72:9706-9713) show that the incorporation of a peptide containing Arg-Gly-Asp (RGD) into the HI loop of the Ad5 fiber loop allowed the resulting recombinant fiber to utilize the RGD-integrin interaction as an alternative infection pathway to CAR.
Einfeld et al. (1999, J. Virol. 73:9130-9136) developed a pseudoreceptor consisting of a membrane-anchored single-chain antibody that recognizes a linear decapeptide from the hemagglutinin (HA) protein. Incorporation of this HA peptide into the HI loop of the Ad2 fiber knob enabled the resulting recombinant adenovirus to transduce pseudoreceptor expressing cells under conditions where CAR binding was blocked.
Xia et al. (2000, J. Virol. 74:11359-11366) screened a nonapeptide phage-display library against the extracellular domain of the human transferrin receptor (hTfR) to identify epitopes specific to the receptor. Two of the sequences identified were inserted within the HI loop of the Ad5 fiber knob, showing proper fiber trimerization and gene transfer to hTfR expressing cells.
Nicklin et al. (2001, Mol. Ther. 4:534-542) demonstrated that two endothelial cell (EC)-binding peptides redirected adenovirus tropism to ECs when inserted within the HI loop of the fiber knob in which CAR binding had been ablated.
The present invention overcomes the limitations of the previous work by expressing a functional Ad5 fiber knob domain on the capsid of bacteriophage λ as a carboxy-terminal fusion to the major head protein D. This phage display system was employed to construct a large collection of peptide sequences incorporated within a functional Ad5 fiber knob wherein the binding to wild-type CAR was ablated. After panning this library on CAR-negative mouse embryo fibroblasts NIH-3T3, three peptide ligands in the knob context were isolated that show binding to CAR-negative cells.
Viruses incorporating these peptides ligands have an enhanced infectivity of CAR-negative cells and cells expressing low levels of CAR. Accordingly, generating recombinant adenoviruses comprising a modified fiber protein incorporating these novel peptide ligands injects a higher activity and functionality into the adenovirus gene therapy/gene vaccination system.